A bacterial metabolic pathway composed of acetate kinase (ACK) and xylulose 5- phosphate/fructose 6-phosphate phosphoketolase (XFP) has been identified in eukaryotic fungi. In bacteria, ACK primarily functions with its partner enzyme phosphotransacetylase (PTA) for the interconversion of acetate and acetyl-CoA. However, PTA is absent in fungi. Instead, ACK forms a putative pathway with XFP, which, like ACK, was also thought to belong almost strictly to the bacteria. ACK-XFP has been proposed to form part of a modified pentose phosphoketolase pathway in the heterofermentative growth of lactic acid bacteria and play a key role in energy generation, but the role of this pathway in fungi has not yet been investigated. Virtually everything known about the enzymology of ACK is from characterization of the E. coli ACK and the only archaeal ACK, that from Methanosarcina. Initial biochemical and kinetic characterization of several eukaryotic ACKs has revealed interesting and unique properties, suggesting that this enzyme family is significantly more diverse than previously thought. These biochemical differences indicate that eukaryotic ACKs may have different physiological functions than their prokaryotic counterparts. Very little is known about the biochemistry and enzymology of XFP. Surprisingly, a number of fungi have two genes encoding this enzyme, raising the possibility that the differences between the encoded enzymes is of biochemical and physiological importance. The objective of this study is to understand the role of the putative ACK-XFP pathway in fungal physiology and metabolism using the genetically tractable basidiomycete Cryptococcus neoformans as the model system. The specific aims of this proposal are two-fold: (1) to investigate the role of the ACK-XFP pathway in fungal metabolism and physiology, and (2) to biochemically and kinetically characterize fungal ACK and XFP enzymes. This will be the first investigation into the physiological role of the ACK-XFP pathway in fungi as well as the first into ACKs that do not primarily partner with PTA. The experiments described in this proposal are expected to provide unique insights into the enzymology and evolution of this important metabolic enzyme as well as contribute to a deeper understanding of the ASKHA (acetate and sugar kinase/Hsc70/actin) superfamily of phosphotransferases. PUBLIC HEALTH RELEVANCE: A bacterial energy generating pathway consisting of the enzymes acetate kinase (ACK) and xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (XFP) has recently been found in a number of medically and economically relevant fungi. If ACK and XFP prove to be essential in fungi, their absence in humans, animals, and plants may allow these enzymes to be investigated as potential targets for development of antifungal agents in the future. However, a thorough understanding of the biochemistry and physiological role of the ACK-XFP pathway in fungi, the objectives of this proposal, is necessary before such endeavors can be considered.